The present invention relates to an improvement in the kind of devices that are being used for detection of fluorescent species.
Fluorescence detection or fluorometry is a well established and often used method within analytical chemistry. The main features of fluorescence detection are high selectivity and very high sensitivity, and the method is often applied to detection of trace constituents in samples of various kinds. Fluorescence detectors consist, in general, of three main subsystems, i/ an excitation light source and associated optics, ii/ a sample cell, and iii/ collection optics and light detector. The light source generates the light that excites the fluorescent species. The most often used light sources are high intensity lamps, like, e.g., xenon lamps or lasers. The excitation optics transports the light from the light source to the illumination zone, where the light excites the sample. Focusing optics is most often used, but also fiber optics and other kinds of waveguides, for example, may be used. When a laser light source is used, the focusing optics may, in some cases, be omitted. The sample can contain one or several fluorescent species. The sample is, in general, present in a medium, e.g., a liquid solution, which in turn is contained in some kind of sample cell. The sample cell may, e.g., be a compartment into which the sample is first loaded, then detected while being. stationary, and finally withdrawn. The cell may also be part of some kind of conduit, through which the sample is transported to and from the illumination zone. The collection optics collects the emitted fluorescent light in an efficient way, and transports it to the light detector. Also for the collection optics, focusing elements are commonly used, but also, e.g., fiber optics may be used. The collection, as well as the excitation, optics may also comprise some kind of device, e.g., a monochromator or one or several filters, for selection or dispersion of wavelengths. The excitation is most often performed at one wavelength or a few well-defined wavelengths, or, alternatively, the excitation wavelength may be scanned. The detection may be performed at one or several discrete wavelengths or wavelength intervals, or scanned or dispersed over a wavelength interval, or the total amount of emitted light may be detected. Wavelength selective detection increases the versatility and selectivity of fluorometry, and is a prerequisite in applications like, e.g., four colour DNA sequencing. There are many different kinds of light detectors, e.g., photodiodes, diode arrays, CTD:s (charge transfer devices, including CCD:s (charge coupled devices) and CID:s (charge injection devices)), and photomultiplier tubes.
One of the most common and most important uses of fluorometry is as a detection method in connection with analytical methods wherein the sample is contained and transported in some kind of conduit. Such analytical methods include, but are not limited to, CE (capillary electrophoresis), LC (liquid chromatography), and FIA (flow injection analysis). In this context, the present invention will mainly be discussed in connection with CE, but applications to other analytical methods are obvious to the skilled person. CE is a well-established separation method with the possibility to analyse very small amounts of sample, and yielding a very high separation efficiency.
Fluorescence detection, and especially LIF (laser induced fluorescence), is a well-established detection technique for CE. Lasers have two main advantages: i/ the high intensity of the light, and ii/ the ability to focus the laser beam to a small spot within the capillary. It is important that the size of the light beam at the point of excitation does not contribute to band broadening: the width of CE peaks may require beam diameters of 100 xcexcm or less. In the most common, and well-established, optical set-up, the orthogonal set-up, the capillary is illuminated with a laser, and the emitted light is collected at 90xc2x0 to the direction of the laser beam. The main concerns, in order to maximise the sensitivity, are to maximise the light collection efficiency, and to minimise the amount of stray light reaching the light detector. High collection efficiency is, in general, obtained by using high numerical aperture collection optics. The term stray light is used here to denote all kinds of unwanted, detected light. Stray light may, to some extent, be rejected through the use of spectral and/or spatial filters. Electrophoresis capillaries are often protected by a polymer coating, e.g., polyimide, which has to be removed before fluorescence detection can be performed. Scattering of primary laser light may occur if there are polymer or other particles left on the capillary wall, if the wall is scratched, or if there are heterogeneities within the wall or the medium inside the capillary. Further, light scattering occurs at every optical interface according to Fresnel""s laws of reflection. In particular, the cylindrical columns ordinarily used in CE pose a problem, since they scatter light also at 90xc2x0 to the direction of the laser beam. Also, most materials scatter light by elastic (Rayleigh) Raman molecular scattering. Scattered primary light may often, but not in all cases, be efficiently rejected by spectral filtering or wavelength dispersion. Wavelength shifted secondary light may present a more severe problem. Inelastic (Stokes shifted) Raman scattering or fluorescence emission from polymer or dirt particles on the column wall, from the column wall itself, from the medium in which the sample is contained, or from impurities in the medium or in the sample itself may not be easily rejected by spectral filtering or wavelength dispersion. Spatial filtering may be obtained by, e.g., shallow focal depth collection optics and apertures. The light collection is spatially concentrated to the region of the medium, while light emanating from other regions is rejected.
For high efficiency separation methods, utilizing small diameter columns and small samples, and yielding very narrow analyte bands at the detector, like e.g., micro-LC and, in particular, CE, it is imperative that the detection is performed on column and that the detection volume is as small as possible. Use of an external detection cell with diameter larger than the column will lead to band broadening, and coupling to such a cell does, in general, cause dead volumes leading to further band broadening. The maximum allowable detection volume for, e.g., a highly efficient CE separation on a 100 xcexcm column may be on the order of or less than 2 nl.
One proposed device for maximising light collection efficiency and minimising stray light is the confocal fluorescence microscope [Ju, J. et al., Anal. Biochem. 1995, 231, 131-40]. A laser beam is reflected by a low-pass dichroic beam splitter, and focused by a microscope objective to a very small spot, on the order of 10 xcexcm, inside the capillary. The emitted fluorescent light is collected by the same objective, but transmitted through the beam splitter to the detection optics. By focusing the collection optics tightly inside the capillary, stray light contributions from the capillary wall are diminished. By placing an aperture at the focal point of the collected fluorescent light, stray light may be further rejected by spatial filtering. High light collection efficiency is achieved by using a high numerical aperture microscope objective. Drawbacks of this device include the need for very strict mechanical tolerances, very careful optical alignment, and the sensitivity to, e.g., vibrations. These drawbacks are a consequence of the shallow focal depth utilized. Further, for cylindrical capillaries, the problem of focusing light and light collection in the interior of a body lacking circular symmetry is encountered.
Another proposed device for optimisation of detection sensitivity is the sheath flow cell [Swerdlow, H. et al., Anal. Chem. 1991, 63, 2835-41; Chen, D. Y. et al., J. Chromatogr. 1991, 559, 237-46]. The analyte to be detected is eluted from the capillary, and excited immediately outside the end of the capillary in a stream of buffer flowing through a high purity quartz cuvette. Since the analyte is detected post-capillary, stray light contributions from the capillary wall are omitted. Further, since the quartz cuvette may be designed with flat optical surfaces, the light scattering problem associated with curved surfaces is omitted. High light collection efficiency is achieved by using high numerical aperture collection optics. The use of this device demands very careful control of flow conditions and flow impedances in order to maintain the integrity of the analyte stream. Further, the presence of particles, bubbles, or impurities in the sheath flow buffer may lead to large amounts of stray light.
In order to increase the sample throughput of CE analysis, like, e.g., for large scale DNA sequencing, it is desirable to run CE in a multitude of capillaries simultaneously. Such multiplexed analysis brings about several additional optical and geometrical problems with regard to fluorescence detection. Most often, the multitude of capillaries are arranged side-by-side in a parallel fashion, so that the array of capillaries form a planar array at the detection point.
The conventional on-column orthogonal set-up may be applied to capillary array detection [Ueno, K. et al., Anal. Chem. 1994, 66, 1424-31; Carrilho, E. et al., Proceedings of the Society of Photo-Optical Instrumentation Engineers 1997, 2985, 4-18]. However, problems with illumination are encountered. If the planar array of capillaries is illuminated by, e.g., a number of parallel laser beams or a line-focused laser beam, the exciting light may form a plane that is orthogonal to the plane of the capillary array. With this geometry, there is no orthogonal direction left for the collection of light. A 90xc2x0 angle between exciting and emitted light may be obtained by tilting the array of capillaries, but such designs lead to the generation of excessive stray light as well as problems with light collection efficiency. As an alternative, the capillary array may be illuminated by one single laser beam in the same plane as the array, which laser beam hits the different capillaries in a subsequent manner [Anazawa, T. et al., Anal. Chem. 1996, 68, 2699-2704; Yeung, E. S. et al., U.S. Pat. No. 5,741,411, 1998]. With this design, the collection optics may be placed at 90xc2x0 to the incoming beam. However, since the incoming beam will hit a multitude of optical interfaces, a lot of stray light is generated. Additionally, since some laser power is lost at each interface, the available laser power will rapidly drop as the laser beam travels through the multitude of capillaries, leading to a decreased fluorescence signal. Further, since laser beams are divergent, it is not possible to keep a tight focus over an extended distance of the beam. The result is that some capillaries will be illuminated by a not so tightly focused beam, which may cause detection band broadening and loss of separation resolution of the electrophoretic peaks.
The principle of the confocal microscope may also be applied to capillary array detection [Mathies, R. A. et al., U.S. Pat. No. 5,274,240, 1993; Kheterpal, I. et al., Electrophoresis, 1996, 17, 1852-59]. In this case, the focused laser beam has to be scanned over the capillary array (or vice versa). Thus, it is necessary to use moving parts in the detector, which is not desirable, especially in view of the demanded tight mechanical tolerances and the susceptibility to vibrations. Further, since the laser power is shared in time between all the different capillaries, the duty cycle per capillary is low, which decreases the total light collection efficiency per capillary. These problems are particularly pronounced when using large arrays of capillaries.
Also the sheath flow cell [Takahashi, S. et al., Anal. Chem. 1994, 66, 1021-26; Dovichi, N. J. et al., U.S. Pat. No. 5,567,294, 1996; Dovichi, N. J. et al., U.S. Pat. No. 5,741,412, 1998; Takahashi, S. et al., U.S. Pat. No. 5,759,374, 1998] may be applied to capillary array detection. However, specific drawbacks are encountered. Again, the simple orthogonal setups discussed above can not be used. One possibility is to illuminate the planar array of analyte streams orthogonally with a plane of light (e.g., a line focused laser beam), and collect the emitted light in the same plane as the capillary array, i.e., end-on collection with respect to the capillaries. However, fundamental optical constraints limit the efficient collection of light from an extended line of objects (i.e., the capillary ends). In order to decrease the longest dimension of the array of capillary ends, an alternative is to arrange the capillaries in a three dimensional array, i.e., in a bundle, but then the laser beam will interact with the samples over an extended distance, which may lead to divergence, loss of focus, and detection band broadening. Also, tight bundling of many capillaries may result in band broadening due to inefficient dissipation of Joule heat. Further, sheath flow detection in connection with capillary arrays put extreme demands on the sophistication, control, and tolerances of the flow system.
A multicapillary DNA sequencing device based on transverse illumination and guiding of the emitted fluorescent light by total internal reflection (TIR) in the capillaries has also been proposed [Takubo, K., JP Patent No. 10019846, 1998]. However, no optical coating on the capillaries was proposed, so TIR conditions will not be fulfilled. Since the refractive index (RI) of the gel inside the capillaries in most practical cases will be approximately equal to the RI of the electrolyte buffer, into which the capillary ends are immersed, most of the light will escape radially through the circumference of the capillaries, and will not reach the capillary ends. Also, bundling of capillaries without optical coating will give rise to severe optical crosstalk between the capillaries, again preventing most of the light from reaching the end of the capillary in which it was emitted. Further, bundling of capillaries all the way from the injection end to the detection point will give rise to substantial Joule heating, impairing the electrophoretic resolution.
Fiber optics may also be used to transport the exciting light and collect the emitted light [Quesada, M. A. et al., Electrophoresis, 1996, 17, 1841-51]. However, alignment of a large number of individual fibers and capillaries involves a huge amount of work. Further, the amount of stray light may be expected to exceed that of the confocal scanner or the sheath flow cell.
The present invention is based on the idea that a device for detection of one or several fluorescent species, said species being contained in a medium, said medium being contained in a conduit, said device comprising a means of exciting the fluorescent species by light, said medium and conduit making up a structure that is transparent to the exciting and the emitted fluorescent light, and said device comprising one or several such structures, may be improved by letting at least part of the emitted fluorescent light be guided away from the illumination zone by total internal reflection (TIR) in said structure and collected from one end of said structure.
Such a device offers simplicity and robustness with respect to mechanics, optics, and liquid handling, as well as high light collection efficiency, low stray light, and easy adaptability to capillary array detection.
For light travelling in a material with refractive index n1 and striking the surface of a material with refractive index n2 at an angle xcex1to the normal to the surface, TIR occurs if
n1 sin xcex1 greater than n2
Thus, n1 has to be larger than n2. Under conditions of TIR, all of the light is, in principle, reflected back into the first material. Fore reflection at angles smaller than xcex1, some of the light is reflected and some is transmitted.
Thus, in one aspect, the present invention provides a device characterized in that the distance between the illumination zone and the light collection end of said structure is large enough to allow light rays emanating from the illumination zone, which do not fulfil the conditions for TIR, to be efficiently transmitted out of the light guiding part of said structure before reaching the light collection end. Such a device ensures that only light guided by TIR through the structure will be collected and detected at the end of the structure. By using a suitable arrangement, most of the exciting, primary light and part of the stray light can be forced not to fulfil the conditions for TIR, and to be transmitted out of the light guiding structure before reaching the light collection end.
In another aspect, the present invention provides a device that is characterized in that the distance between the illumination zone and the light collection end of said structure is at least four times, or preferably at least eight times, or even more preferably at least sixteen times, larger than the largest cross sectional dimension of the light guiding part of said structure. If said distance is four times larger, most of the light that reaches the light collection end will have been subject to at least one reflection event. However, since the rejection of light that is not subject to TIR is more efficient upon multiple reflection events, the values eight or sixteen are more favourable.
Further, if the illumination takes place in the immediate vicinity of the light collection end, scattering and diffraction of the primary exciting light due to edge effects may cause increased levels of stray light. Even, e.g., a well-focused laser beam has a finite extent, and such effects may occur close to sharp edges. The present invention provides for a means of avoiding such effects.
The expression xe2x80x9cspeciesxe2x80x9d is use to denote any fluorescent entity, such as molecules, ions, supra-molecular aggregates, micelles, particles, or whole cells or parts of cells. The expression xe2x80x9cmediumxe2x80x9d is used to denote a liquid of high or low viscosity, a semi-rigid gel, or a solid material. The expression xe2x80x9cconduitxe2x80x9d is used to denote any elongated entity physically containing said medium, in which entity said species may be transported, such as, but not limited to, a tube, a capillary, a column, or a channel formed in, e.g., glass, quartz, silicon, or an organic polymer. In one particular case, the conduit is a separation column for CE or LC. Said medium may or may not be transported within said conduit. The exciting light may be within the ultraviolet, visible, near-infrared or infrared range. The expression xe2x80x9cstructurexe2x80x9d is used to denote any entity comprising and physically defining said conduit and said medium. The term xe2x80x9clight guiding part of the structurexe2x80x9d denotes that part of the structure that is actually guiding the light, and may refer to the medium, the conduit, or the medium and the conduit. The expression xe2x80x9ctransparentxe2x80x9d means that the material must be able to transmit light with low loss, i.e., not highly absorbing and not highly scattering at the relevant wavelengths. The xe2x80x9clight collection end of the structurexe2x80x9d is that end where the guided light rays leave the structure and may be collected and detected by optical means. This mode of light collection excludes the decoupling of light from the structure by means of any external optical decoupler before reaching the end of the structure. An example of such a decoupler may be an optical fiber pigtailed onto the structure. In one particular case, the light collection end is one end of a separation column for CE or LC. The xe2x80x9cillumination zonexe2x80x9d is the location where the exciting, primary light interacts with the structure and excites the fluorescent species.
The advantages of the invention will be better understood from the following discussion of the beneficial influence of different aspects and embodiments of the invention. Clarifying examples will mainly refer to detection in connection with CE, but, as will be apparent to the skilled person, the invention is not limited to such detection.